Phosphatidic acid-comprising compositions

ABSTRACT

PCT No. PCT/IL97/00147 Sec. 371 Date Feb. 24, 1999 Sec. 102(e) Date Feb. 24, 1999 PCT Filed May 6, 1997 PCT Pub. No. WO97/41874 PCT Pub. Date Nov. 13, 1997The invention is to a method for the treatment of withdrawal symptoms selected from the group consisting of nausea, sweating, shaking, substance craving and hot flushes.

This is a 371 of PCT/IL97/00147 filed May 6, 1997.

FIELD OF THE INVENTION

The present invention concerns lipid-based compositions and use of suchcompositions in the treatment of withdrawal syndromes or cancer.

BACKGROUND OF THE INVENTION AND PRIOR ART

Withdrawal syndromes can occur during rehabilitation from addiction todrugs, alcohol, cigarettes and from an abrupt decline in the level ofvarious hormones such as that occurring in menopause women. It isexpressed in symptoms of seizures, sweat, tremor, nausea, depression,increase in rate of heart beat and in blood pressure, and others.Typically such addiction is treated by a "wash out" period in which thedependency is gradually removed with or without drug intervention. Thisprocess is painful and tedious and candidates are thus very oftendiscouraged from entering it. There is thus a strong need for a solutionthat can ease the difficult period of withdrawal and that would allowpeople to return to normal life without much complications.

A basic biochemical phenomenon shared by most of the withdrawalsyndromes is a change in the composition and in the structure ofneuronal cell membranes which is expressed in membrane "fluidity"(Hannan, Am. Rev. Respir. Dis., 140 (1989), 1668-73; Crews,Psycho-pharmacology, 81 (1983), 208-13; Harris, Life Sci., 35 (1984),2601-8; Heron et al., Eur. J. Pharmacol., 83 (1982), 253-261). Thesechanges can at times be counteracted by administration of specialnatural preparations, resulting in the reduction of the symptoms relatedto the withdrawal processes (Heron et al., Eur. J. Pharmacol, 83 (1982)253-261; Shinitzky, Physiology of membrane fluidity, (1984), Vol I,Chapt. 1).

Multi-drug resistance (MDR) is also related to change in the fluidity ofthe cell membrane (Seydel et al. Arch. Pharm., 327. 601-610, 1994). MDRis a major cause of failure of cancer therapy involving use of cytotoxicdrugs, particularly in recurring cancer.

Phosphatidic acid (PA) is a natural phospholipid found in plants andanimal tissues. Its content usually does not exceed 5% of the totalphospholipids in any of these sources. Therefore, lipid extractsavailable for human consumption (e.g., soybean phospholipids) are low inPA. Other sources of processed lipid mixtures for per os consumption orintravenous injection are devoid of PA. These include AL 721 (Antonianet al., Neurosci. Biobehav. Rev. 11 (1987), 399-413); Bros™ (Fidia Sp.A. Abano-Terme, Italy) or Intralipid™ (Vitrum Inc., Stockholm, Sweden).

Enzymatic procedures utilizing the enzyme phospholipase D for thehydrolysis of phospholipids to PA are known (Waite, M. Ed. Thephospholipases, Plenum Press, New York, 1987). However, hitherto no useof such procedures to enrich natural lipase preparations with PA hasbeen made. PA incorporated in a liposome containing phosphatidyl cholinepreparation was shown to reduce toxicity and enhance anti-fungalactivity of the anti-fungal drug Hamycin. It was shown that PA had astrong protective effect and increased the survival of mice by 90% afterseven days of therapy, as compared to mice treated with Hamycin alone(Moonis M. et al., J. Anti. Microb. Chemother., 31:569-579, 1993).Furthermore, a liposome preparation comprising PA was shown to countersymptoms of kidney toxicity caused by amino glucoside antibiotics asdemonstrated by a recuperation in the kidneys' phosphatase activity(Mingert-Leclercq, M. P., et al., Biochem. Pharmacol., 40:489-497,1990).

GLOSSARY

The following is the meaning of some terms of which use will be made inthe text below:

Withdrawal syndrome--a syndrome which is a result of deprivation of thebody of a substance to which the body was exposed continuously over aperiod of time. Such a substance may be an exogenous substance, taken bythe individual, or may be an endogenous one. Exogenous substances mayinclude, for example, drugs of abuse, e.g. heroin, cocaine, morphine,and others, a variety of therapeutic drugs, e.g. sedatives; tobacco,alcohol, and others. Endogenous substances may include a variety ofhormones, particularly such hormones the level of which changes at acertain age. Typical examples of withdrawal syndromes are such thatoccur in addicts during rehabilitation from addiction to drugs, alcohol,tobacco and other exogenous factors. Another example is that occurringin menopausal women, resulting from change in hormonal level during themenopause.

Withdrawal symptoms--symptoms appearing in an individual having awithdrawal syndrome. Examples are nausea, sweating, shaking andsubstance craving, in drug of abuse associated withdrawal syndromes; hotflushes in menopausal women, etc.

PA-enriched lipid preparation (PA-E-LP)--a lipid preparation comprisingat least 10% (w/w) of PA, preferably above about 10% and typicallywithin the range of about 20%-75% of PA, out of the total lipid contentof the composition.

Natural PA-E-LP--a PA-E-LP derived from a natural lipid preparation,e.g. a phospholipid preparation derived from plants, from animal tissue,or any combination thereof. Such a natural phospholipid preparation maytypically be derived from soybean, from egg yolk or from animal sera.The natural PA-E-LP is prepared from a natural phospholipid preparation,typically by an enzymatic process. In a natural PA-E-LP, the remainderof the lipids consist primarily of phospholipids although smallquantities, e.g. 0.1-10%, of other lipophilic substances, such ascholesterol, fatty acids, etc., may also be included in the preparation.

GENERAL DESCRIPTION OF THE INVENTION

In accordance with the invention, a natural PA-E-LP was prepared by anenzymatic process. Such a natural PA-E-LP constitutes one aspect of theinvention.

In accordance with the invention it was also discovered that PA-E-LP,and particularly natural PA-E-LP, is effective in the treatment ofcertain human conditions. It was furthermore surprisingly found inaccordance with the invention, that PA-E-LP, and particularly naturalPA-E-LP is effective in increasing the fluidity of cell membranes andcountering various symptoms associated with or involving changes inmembranes' fluidity, such as withdrawal symptoms or treatment intendedto sensitize MDR resistant cancer cells.

The present invention thus provides by one of its aspects a lipidpreparation derived from a natural source enriched to comprise at leastabout 10% PA, preferably at least about 20% PA and most desirably above50% PA. Typically, the concentration of PA as a total lipid ingredient,would not exceed 75%.

The concentration given above and below as "%" means to denote thenumber of weight units of an ingredient per 100 weight units of theentire composition (w/w).

The natural PA-E-LP is preferably obtained from a natural phospholipidpreparation by enzymatic treatment using a synthetic or natural sourcecomprising the enzyme phospholipase-D. The natural phospholipidpreparation may be of vegetable origin, may be of animal origin, or acombination thereof. Typical examples of natural phospholipidpreparations useful for the preparation of natural PA-E-LP of theinvention are, soya lecithin, egg yolk and phospholipids from animalserum. Examples of phospholipase-D sources, are peanuts, typicallyground peanuts or a phospholipase-D fraction derived therefrom.Phospholipase-D or a phospholipase-D source is added in an amount andfor a time sufficient to hydrolyze at least about 10% or preferablyabout 25% and most preferably about 50% of the phospholipids to yieldPA-E-LP.

By another aspect of the present invention there are providedpharmaceutical compositions comprising PA-E-LP as the active ingredient.Particularly preferred are such compositions comprising natural PA-E-LPas the active ingredient. Examples are pharmaceutical compositions foruse in reducing withdrawal symptoms; pharmaceutical compositions for usewithin the framework of cancer treatment to increase sensitivity ofcancer cells, particularly such cells possessing MDR properties, tocytotoxic drugs; etc.

Also provided in accordance with the invention is a method for thetreatment of a human condition or disease comprising administering to apatient in need, an effective amount of PA-E-LP.

The term "effective amount" should be understood as an amount of aningredient sufficient to yield a desired therapeutic effect. Forexample, within the framework of the treatment of withdrawal syndromes,an effective amount will be an amount which gives rise to permanent ortemporary alleviation of withdrawal symptoms in individual, alleviationbeing either reduction of intensity of withdrawal symptoms or reductionin the rate of appearance of such symptoms, or generally improvement inany parameter characteristic of such withdrawal symptoms. In the case ofanti-cancer treatment, an effective amount is an amount giving rise toincreased sensitivity of the tumor cells toward cytotoxic drugs, asdetermined for example by a decrease in the size of a tumor mass.

The pharmaceutical composition of the invention may typically be orallyadministered although it may also be formulated for topical orparenteral administration. For oral administration the composition maycomprise various flavoring agents, edible colors, etc. Furthermore thecomposition for oral use may also be encapsulated, e.g. in anenterocoated gelatine capsule.

For parenteral administration the composition will typically be injectedintravenously (I.V.). Such a parenteral composition may for examplecomprise soybean multi-triglycerides, egg phospholipids, PA prepared inaccordance with the invention, glycerol and distilled water.

A topical composition may be in the form of a gel or a salve and maythus comprise various additives known per se to allow the compositionsto obtain such a physical form (e.g. a gelating agent).

The compositions may be used for the treatment of a variety of humanconditions and diseases. One preferred embodiment of the invention isconcerned with the treatment of withdrawal syndromes. The treatment maybe manifested by a permanent improvement in an individual's condition,e.g. the disappearance or reduction of intensity of the withdrawalsymptoms, or a temporary disappearance or reduction of such symptomsduring an attack.

Withdrawal syndromes which can be treated in accordance with theinvention include treatment of menopausal women, particularly treatingsuch women to reduce the occurrence of hot-flushes during the menopausalperiod; treatment of withdrawal syndrome during rehabilitation fromsmoking or drug or alcohol addiction; etc.

Another preferred embodiment of the invention is concerned with thetreatment of cancer, particularly such related with acquired multi-drugresistance MDR. Development of MDR is a reaction of tumor cells tocontinuous exposure to cytotoxic agents both in vitro and in vitro. Thismechanism is believed to be responsible for the resistance developed bycancer patients to chemotherapy treatment which is currently one of themost serious problems in cancer treatment. It has been shown that thesensitivity of tumor cells to various cytotoxic drugs is correlated withtheir potency for membrane fluidization. Treatment with the compositionof the invention may increase the fluidity of the membrane of tumorcells present in an individual, thereby reversing their acquiredmulti-drug resistance to various cytotoxic drugs and thus enablingcontinuation of a chemotherapy regimen, which may result in theimprovement of the treated individual's condition.

The invention will now be illustrated in the following examples withoccasional reference to the annexed drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation depicting the preparation of the PAenriched mixture of the invention. As can be seen in the figure, theratio of the starting materials (peanuts, soya lecithin and water) is1:1:7.5, respectively.

FIG. 2 is a schematic representation depicting the separation of thelipid phase of the PA-mixture prepared by the reaction showed in FIG. 1.

FIG. 3 is a schematic representation depicting the preparation of thePA-mixture of the invention prepared as described in FIGS. 1 and 2 in asuitable form for filling containers.

FIG. 4 is a schematic representation depicting the preparation oftablets from the PA-mixture prepared as described in FIGS. 1 and 2.

FIG. 5 shows the formation profile of PA during an enzymatic hydrolysisof soybean phospholipids. (The results are present as % from the entirecomposition).

FIG. 6 shows the effect of PA on lipid viscosity of human lymphocytes.The results are presented as the degree of fluorescence polarization Pof DPH in the membranes of the washed lymphocytes and are translated to2P/(0.46-P), which is a linear scale of lipid viscosity (Shinitzky andBarenholtz, Biochim, Biophys. Acta. 515, (1978), 367-394).(PC--phosphatidylcholine from egg yolk; AL721--a mixture oftriglycerides, PC and phosphatidylethanolamino (PE) from egg yolk;PA--phosphatidic acid obtained by enzymatic reaction of soybeanphospholipids; PA+egg PC:1:1--a mixture of PA and PC in equalproportions (by weight)).

FIG. 7 is a graphic representation depicting the mean number ofcigarettes smoked by subjects participating in a study assessing theaffect of the PA-mixture of the invention on smoking withdrawal symptoms(See Example 5). The number of cigarettes smoked per day by subjects ofthe treatment group (treated with the PA-mixture of the invention) wascompared to the number of cigarettes smoked by subjects of the controlgroup (receiving a non-active mixture) at different time periods afterthe beginning of the study. The number of cigarettes is expressed by amean score calculated as explained in Example 5.

FIG. 8 is a graphic representation showing the need to smoke of subjectsparticipating in a study assessing the affect of the PA-mixture of theinvention on smoking withdrawl symptoms (Example 5). The need to smokeof subjects in the treatment group (receiving daily treatment with thePA-mixture of the invention) was compared to that of subjects of thecontrol group (receiving non-active mixtures daily) at different timesafter the beginning of the study.

EXAMPLE 1 Preparation of PA Mixture

(a) Preparation of a Reaction Product

FIG. 1 schematically depicts the method of preparation of a PA mixturein accordance with the invention. As can be seen, the starting materialsare the following (the amounts calculated in the Example are for a 11mixture):

1. 150 grams of grained fresh peanuts as an origin of Phospholipase D.

2. 150 grams of granulated soy lecithin as an origin of soybeanphospholipids.

3. 1000 ml. to be added to H₂ O.

The above-mentioned starting materials were mixed in a meat blender,brought to homogenization and then the pH of the mixture was adjusted toa pH of 5.3-5.4 by the addition of calcium lactate, sorbate, benzoate,ascorbic acid, citric acid and an anti-oxidant (each in the amountdescribed in the figure). The mixture was then reacted under continuousmixing for four hours at 36-38° C. after which the pH was once moreadjusted to a pH of 3.9 by the addition of citric acid and sugar inamounts described in the figure. The mixture was then stored at 4° C.overnight.

(b) Separation of the Lipid Fraction

The reaction mixture obtained by the above procedure was then furtherseparated by a procedure depicted schematically in FIG. 2. As seen inthe figure, the reaction product was subjected to three main separations(indicated as separations I, II and III in the figure) wherein,generally, in each separation the aqueous phase of the mixture wasremoved from the bottom until, finally, the separated lipid phase wascollected and stored in a freezer (-18° C.).

(c) Preparation of the Lipid Composition for Filling Containers

The lipid mixture obtained by the above separation was subjected toseveral additional procedures by which it was prepared for fillingcontainers. As seen in FIG. 3, the lipid mixture obtained by the aboveseparation process was first thawed and then treated by heating,cooling, homogenization and mixing as described in FIG. 3 to form acomposition suitable for filling containers. The containers are thenstored at 4° C. Typically, the size of the containers is such whichaccommodates half a liter of the mixture.

(d) Preparation of PA Containing Tables

The PA mixture obtained by the method described in Examples 1(a)-1(c)above and shown in FIGS. 1-3 may also be processed into tablets. Themethod for preparation of tablets comprising the PA mixture is shown inFIG. 4. As seen in the figure, generally the phospholipid paste obtainedby the above process was first defrosted and then heated and the pH ofthe mixture adjusted to a high pH of about 9. Following addition of zincacetate to the mixture a white sediment appeared which, after washingand drying, was formulated into tablets.

(e) Profile of PA formation

The profile of formation of PA is depicted in FIG. 5. As can be seen,after about 24 hours, more than 80% of the soybean phospholipid washydrolyzed yielding a PA enriched preparation.

EXAMPLE 2 The effect of Phosphatidic Acid (PA) on Lipid Viscosity ofHuman Lymphocyte Membranes

Human lymphocytes (3×10⁶ /ml) from 10 healthy individuals were incubatedwith 1 mg/ml of 4 different phospholipid preparations and 1 controlpreparation (phosphate buffered saline--PBS).

The results depicted in FIG. 6 demonstrate the effect of PA indecreasing membrane viscosity (i.e. increasing cell membrane fluidity).It can be seen that at all times, the effect of PA above or in a 1:1mixture with PC was much stronger than the two other testedpreparations.

EXAMPLE 3 Treatment of Menopause Women With a 50% Lipid Mixture

Three groups, each consisting of 10 women over 50 who suffered from hotflushes syndrome, participated in the study. Group A continued routinelife without treatment. Group B consumed 12 g of 50% PA lipid mixture(prepared as in Example 1) dispersed in 50 ml of water before breakfastfor 40 days. Group C similarly received 24 g of the 50% PA rich lipidmixture in 100 ml of water for 40 days. Each of the participants inGroup A, B and C was asked to record the number and intensity level oftheir daily hot flushes from day 3 to day 40 of the experiment.Participants of Group C were asked to determine these parameters for 3days one month after termination of the trial (Group D). The results arepresented in Table 1 below:

                  TABLE 1                                                         ______________________________________                                        Daily number of mild, moderate and severe levels of                             hot flushes (average ± S.D.):                                              Group   Mild     Moderate                                                                             Severe Total  Weighted                              ______________________________________                                        A     0.75 ± 0.9                                                                          5.0 ± 4.7                                                                           10.5 ± 3.1                                                                        16.3 ± 6.2                                                                        42.3 ± 15.5                            B 3.50 ± 3.3 2.5 ± 4.3  2.5 ± 4.3  8.5 ± 2.3 16.0 ± 9.0        C 3.50 ± 2.5 1.5 ± 0.7  2.3 ± 3.8  7.3 ± 1.0 13.4 ± 10.0       D 2.50 ± 3.7 4.8 ± 5.5  4.2 ± 5.6 11.5 ± 2.4 24.7 ±          ______________________________________                                                                              11.0                                     A  Control                                                                    B  12 g of lipid formulation per day                                          C  24 g of lipid formulation per day                                          D  One month after termination of medication in Group C.                 

EXAMPLE 4 Alleviation of Morphine Withdrawal Symptoms by 50% PA RichLipid Mixture

Experiments were carried out as outlined in Heron et al., Eup. J.Pharmacol., 83, (1982), 253-261. Two groups of 20 mice each wereinjected subcutaneously twice daily with morphine HCl solution (10mg/ml) in saline up to 100 mg/kg for 9 days, or with saline alone(control). The treated group was divided into two sub-groups eachreceiving a 6% lipid diet. One group of mice received corn oil and theother a PA rich diet (the PA preparation prepared as in Example 1). Thewithdrawal syndromes were induced by injection (i.p.) with naloxone (2.5mg/kg), and then recorded for a period of 25 min in a blind mode.

The results, shown in Table 2 below, demonstrate that PA had a verystrong reducing effect on the mices' withdrawal symptoms.

                  TABLE 2                                                         ______________________________________                                        Alleviation of morphine withdrawal symptoms                                     by 50% PA rich lipid mixture                                                                  Purina-chaw Purina-chaw                                        diet + 6% corn oil diet + 6% PA lipid                                        Symptoms (control group) mixture                                            ______________________________________                                        1. Jumps      32 ± 10  5 ± 0.6                                            2. Body shakes 15 ± 4  6 ± 2                                            3. Forelimb tremor <50% >20%                                                ______________________________________                                         Jumps  Number of jumps per 25 min.                                            Body shakes  Number of body shakes per 25 min.                                Forelimb tremor  Percent of animals showing continuous and strong tremor      of forelimbs (more than 70 episodes in 25 min). Control groups were void      of any behavioral abnormalities.                                         

EXAMPLE 5 Alleviation of Smoking Withdrawal Symptoms by 50% PA EnrichedLipid Mixture

37 chronic smokers of 20-40 cigarettes per day (CPD) formed theexperimental group for testing the effect of the PA lipid mixture of theinvention on withdrawal symptoms associated with smoking cessation. Theprerequisites for admission to the experiment were:

(a) willingness of the subject to stop cigarette smoking;

(b) self-confessed inability to stop smoking without externalassistance.

Initially, the subjects volunteered for the study were divided at randominto two equal groups subjects of the first group receiving a one dailydose of the 50% PA mixture (prepared as described in Example 1 above)and subjects of the second group (control group) receiving a one dailydose of a non-active preparation. However, due to abandonment of severalof the subjects at the beginning of the study as well as the strikingoff of results of several other subjects who completed the study but didnot diminish their rate of smoking at all, the remaining 37 subjectswere divided into the two above groups as shown in Table 3 below:

                  TABLE 3                                                         ______________________________________                                        PA-preparation   Non-active preparation                                                                      Total                                          ______________________________________                                        Male    8            13            21                                           Female 10  6 16                                                               Total 18  19  37                                                            ______________________________________                                    

The subjects received either the PA mixture or the non-active mixtureover a period of one month.

The following six parameters were studied throughout the study:

(a) parameters for evaluating smoking heaviness

(i) number of cigarettes smoked per day;

(ii) time between waking and smoking the first cigarette; and

(iii) difficulty of refraining from smoking in public places.

The above three parameters were tested at day 0 of the study, at day 7and at day 21 of the study.

(b) Parameters for evaluating the severity of withdrawal symptoms

(i) disquiet;

(ii) extra appetite; and

(iii) need to smoke.

The above parameters were tested at day 7 of the study and on day 21 ofthe study. Various blood parameters of each of the subjects were alsoevaluated (including a complete blood count evaluation of subpopulations of T-cell, B-cell and NK cell numbers and evaluation ofintracellular cytokines) at day 0 of the study as well as at the end ofthree weeks of the study.

All the parameters were scored on a 0-4 scale (expressed finally on ascale of 1-5 respectively since some tests did not accept 0 as aparameter).

By using logistic regression statistical analysis, it was decided thatthe indicative parameters in the test would be the number of cigarettessmoked by a subject per day at the end of week three (day 21) of thestudy and the desire to smoke expressed by the same subject on the sameday. A comparison was made to assess whether subjects treated by thePA-mixture smoked less at the end of week three of the experiment ascompared to subjects of the control group receiving the non-activemixture and whether subjects of the above first group also had lesscraving for smoking at the end of week 3 of the study as compared tosubjects of the above control group.

Results

Statistical analysis of the results using Students Test andMann-Whitney's U test of the above study showed the following:

1. As seen in FIG. 7, the number of cigarettes smoked per day at the endof week 3 of the study was significantly smaller in subjects of thegroup treated with the PA-mixture as compared to that of subjects of thecontrol group (α<0.05).

2. Statistical analysis showed that due to the fact that the tendenciesof the unrelated tested samples were non-normal and the variances of thetwo compared groups was unequal, the statistical analysis should beperformed by Kolmogorov-Smnirnov's test. According to this test, and asseen in FIG. 8, the desire of subjects of the group grated by thePA-mixture at the end of three weeks of the study is significantlysmaller than that of subjects of the control group (α smaller than0.05).

EXAMPLE 6 Alleviation of Alcohol Withdrawal Symptoms by 50% PA RichLipid Mixture

Volunteers for this study are alcohol addicts at the age of 25-65 whichare defined in the clinic as "alcoholics" which frequently become"drunks". The above two terms "alcoholics" and "drunks" are used inaccordance with the definitions provided by the World HealthOrganization 1951 which are used until today (Jelinek, E. M., TheDisease Concept of Alcoholism" New Haven, Hill House Press, 1960 andHamburg S., "Alcohol Withdrawal in Family and Community" (In Hebrew),1976, respectively).

The following parameters are tested at the beginning of the study and atits end:

(a) Independent parameters

(i) personal data;

(ii) demographic, socio-economic and health data: and

(iii) characterization of addiction habits (quantitative andqualitative).

Suitable volunteers are such that are addicted to alcohol (which is nottable wine or beer) and which have expressed their desire to withdrawfrom drinking. Subjects are people who are active at work, do not sufferfrom other chronic illnesses or from neurological or psychiatricdisorders besides the addiction. In addition, subjects are not addictedto drugs and do not take psychiatric medicines regularly. Furthermore,all subjects are people that have a supportive family that can also bequestioned as to the changes which the subject undergoes during thestudy.

(b) Dependent parameters

(i) physiological changes: pulse, blood pressure, sensitivity tovibrations, capability of carrying out gentle actions, overview ofcalorie intake which is not from alcohol;

(ii) level of functioning in accordance with the following parameters:

(a) home and social functioning hours;

(b) hours in which subject is disconnected homewise and socially;

(c) coping with financial matters;

(d) quality of partnership in a family load.

(iii) an effective evaluation is carried out with the aid of aquestionnaire before and after treatment.

Subjects in the study are divided into two groups:

(1) subjects receiving a syrup comprising the PA-mixture of theinvention twice a day for a month;

(2) subjects receiving the syrup without the PA-mixture twice a day fora month.

The above-mentioned dependent and independent parameters of the subjectsof above group (a) (treatment group) are compared to the same parametersof subjects of the above group (b) (control group) at the beginning ofthe study and at its end (after one month).

EXAMPLE 7 Membrane Fluidity of Drug Resistance Cell Lines Treated WithPhospholipid Acid (PA) and Soy Phospholipids (SP)

Resistant cell lines were developed as described by Ramu et al. (CancerChemother. Pharmacol., 515, 367-394, 1978). P388 murine leukemia cells(Ramu A. et al. Cancer Res., 43, 5533-7, 1983) were cultured in thepresence of increasing amounts of methotroxeate (MET) and doxorubicine(DOX) up to a concentration of 10⁻⁶ M. The resulting cell lines weredesignated P388/MET and P388/DOX respectively.

Cells of the above resistant cell lines were then treated with thefollowing lipid dispersions in saline for 1 hour at room temperature:soy phospholipids (SP); soy phospholipids converted by phospholipase Dtreatment to >90% phosphatidic acid (PA) and a 1:1 mixture (w/w) ofthese preparations. The results (shown in Table 4 below) are presentedin fluorescence polarization values (P) of 1,3,5 diphenyl hexatriene(DPH) or by the linear scale 2P/(0.46-P) as described in Shinitzki etal. Biochem. Biophys. Acta. 515, 367-394, 1978.

                  TABLE 4                                                         ______________________________________                                        Cells              P (25° C.)*                                                                        2P(0.46-P)                                     ______________________________________                                        P388 (unadapted)   0.225 ± 0.015                                                                          1.9                                              P388/MET 0.280 ± 0.020 3.1                                                 P/388/DOX 0.285 ± 0.020 3.3                                                P388/MET treated with SP 0.275 ± 0.030 3.0                                 P388/MET treated with SP 0.235 ± 0.040 2.1                                 P388/MET treated with PA + SP 1:1 0.245 ± 0.035 2.3                        P388/DOX treated with SP 0.275 ± 0.040 3.0                                 P388/DOX treated with PA 0.235 ± 0.025 2.1                                 P388/DOX treated with PA + SP 1:1 0.238 ± 0.025 2.2                      ______________________________________                                         *Values are given as mean ± S.D. of 8 different experiments.          

The results clearly show that, as previously shown, drug-resistantadapted P388 cells acquire a more rigid membrane than non-treated cells.A short exposure to fluidizing lipids (1 hour), which is relevant fordietary treatment in cancer patients, had only a little effect incombination with SP while treatment of the cells in accordance with theinvention with PA and PA+SP was more effective and restored the membranefluidity almost to the level of unadapted non drug-resistant cells.

EXAMPLE 8 Effect of Treatment With PA or SP on Growth Rates of P388Cells.

P388/MET and P388/DOX resistant cell lines were developed as describedin Example 7 above. Non-treated P388 cells and resistant cell lines ofthese cells were grown in the presence and absence of 10⁻⁸ M of MET orDOX as indicated in Table 5 below. The growth rate of each of the abovecells was monitored and is shown in the Table.

                  TABLE 5                                                         ______________________________________                                        Cells                   relative growth rate*                                 ______________________________________                                        P388 (unadapted)        100                                                     P388 grown in the presence of MET 10.sup.-8 M 10 ± 5                       P388 grown in the presence of DOX 10.sup.-8 M 10 ± 5                       P388/MET 100                                                                  P388/MET grown in the presence of 10.sup.-8 MET 95 ± 5                     P388/MET grown in the presence of 10.sup.-8 DOX 95 ± 5                     P388/MET treated with 1 mg/ml SP for 1 hour and 85 ± 5                     then grown in the presence of 10.sup.-8 M MET                                 P388/MET treated with 1 mg/ml PA for 1 hour and 20 ± 5                     then grown in the presence of 10.sup.-8 M MET                                 P388/MET treated with 1 mg/ml PA + SP for 1 hour 25 ± 5                    and then grown in the presence of 10.sup.-8 M MET                             P388/MET treated with 1 mg/ml SP for 1 hour and 90 ± 5                     then grown in the presence of 10.sup.-8 M DOX                                 P388/MET treated with 1 mg/ml PA for 1 hour and 20 ± 5                     then grown in the presence of 10.sup.-8 M DOX                                 P388/MET treated with 1 mg/ml PA + SP 1:1 for 1 30 ± 5                     hour and then grown in the presence of 10.sup.-8 M DOX                        P388/DOX 100                                                                  P388/DOX grown in the presence of 10.sup.-8 DOX 95 ± 5                     P388/DOX grown in the presence of 10.sup.-8 MET 93 ± 5                     P388/DOX treated with 1 mg/ml SP for 1 hour and 85 ± 5                     then grown in the presence of 10.sup.-8 M DOX                                 P388/DOX treated with 1 mg/ml PA for 1 hour and 20 ± 5                     then grown in the presence of 10.sup.-8 M DOX                                 P388/DOX treated with 1 mg/ml PA + SP 1:1 for 1 24 ± 5                     hour and then grown in the presence of 10.sup.-8 M DOX                        P388/DOX treated with 1 mg/ml SP for 1 hour and 90 ± 5                     then grown in the presence of 10.sup.-8 M MET                                 P388/DOX treated with 1 mg/ml PA for 1 hour and 20 ± 5                     then grown in the presence of 10.sup.-8 M MET                                 P388/DOX treated with 1 mg/ml PA + SP 1:1 for 1 28 ± 5                     hour and then grown in the presence of 10.sup.-8 M MET                      ______________________________________                                         *relative growth rate = (percentage growth rate ÷ growth rate of          unadapted P388 cells) × 100                                        

As clearly shown in this Example, treatment of adapted drug-resistantP388 cells with PA or PA+SP 1:1 restore their sensitivity to either METor DOX while SP treatment alone, on the other hand, had only a smalleffect on the cells. It should be noted that the resistance developed inP388/MET cells is also applicable to DOX and vice versa. Similarly, therestoration of susceptibility to drug treatment by PA or PA+SP inP388/MET or P388/DOX cells is non-specific and is applicable to bothdrugs.

What is claimed is:
 1. A method for the treatment of withdrawal symptomsselected from the group consisting of nausea, sweating, shaking,substance craving, and hot flushes in humans, comprising administeringto the patient an effective amount of a lipid preparation comprising atleast about 10% (w/w) of phosphatidic acid (PA).
 2. The method accordingto claim 1, wherein the treatment is for withdrawal symptoms associatedwith smoking cessation.
 3. The method according to claim 1, wherein thetreatment is for withdrawal symptoms occuring during rehabilitation fromdrug or alcohol addiction.
 4. The method according to claim 1, whereinthe treatment is for withdrawal symptoms occuring in menopausal women.5. The method according to claim 4, wherein the withdrawal symptoms arehot flushes occurring during menopause.